Okamoto Yu, Nishimura Naoko, Matsuda Kazunari, Ranawakage Deshani C, Kamachi Yusuke, Kondoh Hisato, Uchikawa Masanori
Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
School of Environmental Science and Engineering, Kochi University of Technology, Kochi, Japan.
Dev Growth Differ. 2018 Apr;60(3):133-145. doi: 10.1111/dgd.12427. Epub 2018 Mar 8.
To elucidate the transcriptional regulation that underlies specification of the otic placode, we investigated the Sox3 downstream enhancer Otic1 of the chicken, the activity of which is restricted to and distributed across the entire otic placode. The 181-bp Otic1 enhancer sequence was dissected into a 68-bp minimal activating sequence, which exhibited dimer enhancer activity in the otic placode and cephalic neural crest, and this was further reduced to a 25-bp Otic1 core sequence, which also showed octamer enhancer activity in the same regions. The Otic1 core octamer was activated by the combined action of Sall4 and the SoxE transcription factors (TFs) Sox8 or Sox9. Binding of Sall4, Sox8 and Sox9 to the Otic1 sequence in embryonic tissues was confirmed by ChIP-qPCR analysis. The core-adjoining 3' side sequences of Otic1 augmented its enhancer activity, while inclusion of the CAGGTG sequence in the immediate 3' end of the 68-bp sequence repressed its enhancer activity outside the otic placode. The CAGGTG sequence likely serves as the binding sites of the repressor TFs δEF1 (Zeb1), Sip1 (Zeb2), and Snail2, all of which are expressed in the cephalic neural crest but not in the otic placode. Therefore, the combination of Sall4-Sox8-dependent activation and CAGGTG sequence-dependent repression determines otic placode development. Although the Otic1 sequence is not conserved in mammals or fishes, the activation mechanism is, as Otic1 was also activated in otic placode tissues developed from mouse embryonic stem cells and transient transgenic zebrafish embryos.
为了阐明耳基板形成过程中的转录调控机制，我们研究了鸡的Sox3下游增强子Otic1，其活性局限于整个耳基板并在其中分布。181 bp的Otic1增强子序列被解析为一个68 bp的最小激活序列，该序列在耳基板和头部神经嵴中表现出二聚体增强子活性，进一步缩减为一个25 bp的Otic1核心序列，其在相同区域也表现出八聚体增强子活性。Otic1核心八聚体通过Sall4与SoxE转录因子（TFs）Sox8或Sox9的联合作用而被激活。通过染色质免疫沉淀定量PCR（ChIP-qPCR）分析证实了Sall4、Sox8和Sox9在胚胎组织中与Otic1序列的结合。Otic1核心相邻的3'端序列增强了其增强子活性，而在68 bp序列紧邻的3'端包含CAGGTG序列则会抑制其在耳基板外的增强子活性。CAGGTG序列可能作为阻遏转录因子δEF1（Zeb1）、Sip1（Zeb2）和Snail2的结合位点，这些因子均在头部神经嵴中表达，但不在耳基板中表达。因此，Sall4-Sox8依赖的激活与CAGGTG序列依赖的抑制相结合决定了耳基板的发育。尽管Otic1序列在哺乳动物或鱼类中不保守，但激活机制是保守的，因为Otic1在从小鼠胚胎干细胞发育而来的耳基板组织和瞬时转基因斑马鱼胚胎中也被激活。
